Project Summary/Abstract The adaptive immune system is a powerful means by which higher organisms protect themselves from a variety of pathogens. T-helper 17 (TH17) cells are an important component of the adaptive immune system involved in the production of inflammatory cytokines responsible for this protection. Dysregulation of inflammatory cytokines causes tissue destruction and chronic inflammation in diseases such as rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease. The TH17-specific nuclear receptor ROR?t is a key transcriptional regulator of these cytokines. Although the upstream regulation of ROR?t had remained elusive, recent studies have revealed a non-coding RNA (ncRNA) and a protein as regulators of ROR?t. The objective of this proposal is to understand the molecular interactions between these key regulatory players, as well as the mechanism of cytokine transcription activation. The hypothesis is that formation of the ternary complex induces a conformational rearrangement in ROR?t which facilitates transcription. To test this hypothesis, it is proposed to identify the interactions and characterize biochemically and biophysically the conformational re-arrangements in the ternary complex between the protein co-regulator, ncRNA, and ROR?t. Specific Aim 1 is devoted to understanding the molecular features of the RNA and both proteins involved in forming interactions. These interactions will be identified through RNA and protein probing and footprinting and other techniques in solution. Furthermore, mutations in the ncRNA found to negatively affect ROR?t regulation will be probed through footprinting to gain insights into transcriptional regulation. Specific Aim 2 will elucidate the intermolecular interactions required for ROR?t binding to promoter DNA and the structural features of the ternary or quaternary complex formation through structural determination by X-ray crystallography, cryo- electron microscopy, and other methods. Finally, Aim 3 focuses on understanding the molecular mechanism of transcriptional regulation elicited by the complex by testing mutations at the molecular interfaces in a reporter system using TH17 tissue culture cells. The proposed study will reveal the molecular interactions involved in ROR?t regulation and cytokine gene production. This proposal is relevant to public health since it addresses the molecular basis of cytokine production in inflammatory autoimmune disorders and other diseases. Understanding the molecular interactions involved between ROR?t and co-activators will aid the development of novel therapeutic drugs. Thus, the proposed research aligns closely with the NIH mission to expand fundamental scientific knowledge in order to prevent and cure diseases.